To what extent changes in gene expression regulation drive the evolutionary innovations of life is an important unresolved question. In particular, the role of cis-regulation has been contentious. While some contend that changes in cis-regulation are responsible for the majority of morphological adaptations, others point out that only a few such cases have been demonstrated. The paucity of examples of adaptive regulatory divergence (cis-acting or otherwise) may be due in large part to the fact that no method for identifying such cases from genome-scale data has yet been developed. We have recently developed the first such method, which is based on analysis of genome-scale catalogs of regulatory differences between any pair of strains or species. We have demonstrated its ability to detect genes and even entire functional groups/pathways that have been subject to positive selection for changes in gene expression. What is now most needed is additional data from which to detect the signature of positive selection, as well as methods to pinpoint and functionally characterize the individual nucleotide changes responsible for these adaptations. Saccharomyces budding yeast represents an ideal model in which to study these questions. The end result of this work, in addition to a greatly increased understanding of how regulatory evolution occurs at the molecular level, will be a comprehensive catalog of cis-regulatory changes in yeast, as well as the first collection of strains (from any species) differing only by single adaptive mutations. We believe that this collection of results and resources will transform yeast into the leading model organism for studying gene expression adaptation. PUBLIC HEALTH RELEVANCE: The subject of this project-the evolution of gene expression-is of great importance to biomedicine. For example, a general understanding of mechanisms of gene expression evolution could be applied to pathogens to help understand the rapid emergence of drug resistant strains and other evolutionary dynamics that occur at scales ranging from a single host infection to global epidemics. Indeed, one yeast strain we propose to study (YJM789) is pathogenic in immunocompromised humans. In addition, a deeper understanding of human gene expression evolution will be essential for elucidating the molecular mechanisms underlying human-specific phenotypes, including many disease phenotypes.